The proposed multi-disciplinary research combines contemporary synthetic peptide/protein chemistry, structural biology, cancer biology, and various biochemical and biophysical techniques, showcasing a powerful, innovative, and integrated approach to filling an important gap in peptide-based anticancer drug discovery. If funded, this project will lead to the addition of new weapons to the existing anticancer arsenal, and broadly impact the development of peptide therapeutics for targeted molecular therapy of many other human diseases as well. Background: p53 is activated as a transcription factor to induce powerful growth inhibitory and apoptotic responses to cellular stress, but is otherwise tightly controlled in normal cells by its negative regulators MDM2 and MDMX. Impairment of the p53 pathway is a hallmark of almost all human tumors where either the TP53 gene is mutated or the p53 protein is functionally inactivated by MDM2 and MDMX. Over-expression or amplification of MDM2 and MDMX in many tumors correlates with a normal (wild type) status of p53, contributing to robust p53 inhibition and degradation. Recent studies have validated inhibition of the p53-MDM2/MDMX interaction as a new therapeutic paradigm for cancer treatment. Our laboratory has developed a series of high-affinity D- peptide antagonists of MDM2 and MDMX (DPMI-?,?,?,?) that, when delivered via a liposomal carrier vehicle, are capable of killing tumor cells in vitro and in vivo by activating the p53 pathway. Objective: Using DPMI-? as a parent molecule (KD = 220 pM for MDM2 and 200 nM for MDMX), we seek to develop high-affinity, protease-resistant, and cell-penetrating D-peptide antagonists of MDM2 and MDMX for potential anticancer therapy. Specific Aims: (1) Design side-chain cross- linked forms of DPMI-? capable of traversing the cell membrane to stabilize intracellular p53. We will use the hydrocarbon stapling technique to design a series of side-chain cross-linked analogs of DPMI- ?, and evaluate their functional and structural properties with respect to ?-helicity, MDM2/MDMX binding, and membrane permeabilization. (2) Test the hypothesis that high-affinity, protease- resistant, and cell-penetrating D-peptide antagonists of MDM2 and MDMX reactivate the p53 pathway and kill tumor cells through cell cycle arrest and/or apoptosis induction. We will evaluate tumor-killing activity of side-chain cross-linked forms of DPMI-? using a variety of tumor cell lines with or without wild type p53. Our long-term objective is to develop D-peptide-based p53 activators as a novel class of anticancer therapeutics for clinical use.